Project Summary: This multidisciplinary proposal aims to define how proanthocyanidins (PACs), a major class of grape polyphenols (GPs), alter the intestinal milieu to promote a biological signature associated with resilience to metabolic syndrome (MetS) and type-2 diabetes (T2D). Using urine, blood, and gut microbiota samples from PAC-supplemented mice and GP-supplemented humans, microbiome-wide association studies will be performed to correlate changes in bacterial strains/species to increases/decreases in microbial metabolites (MMs). PAC treatment of bacteria in vitro and of germfree mice inoculated with bacterial isolates or defined consortia will establish relationships between specific commensal bacteria and PAC-derived MMs, which will then be tested for bioactivity in mammalian cell culture assays and a high-fat diet (HFD)-induced mono- associated germfree (GF) model of MetS/T2D. PACs are associated with metabolic resilience; however, mechanism(s) of systemic protection have remained elusive due to generally poor absorption and uncertainty about molecular targets. PACs reach the colon where they are biotransformed to MM with greater bioavailability; however, the specific bacteria responsible for these transformations, the molecular targets of resulting MM, and validation of their efficacy in preclinical models of MetS/T2D remain to be investigated. We observed that GP supplementation can induce a bloom in the mucolytic gut bacterium Akkermansia muciniphila in association with reduced serum lipopolysaccharide, less intestinal and systemic inflammation, increased expression of tight junction protein occludin, improved glucose metabolism, and less adiposity and weight gain in HFD-fed mice. Increased abundance of A. muciniphila has been observed after gastric bypass surgery and metformin treatment, underlining its importance in positive metabolic outcomes. GP-supplemented mice also showed: 1) decreased bacterial community richness; 2) alterations in genera consistent with improved gut barrier integrity and lactic acid-production; 3) decreased serum levels of bacterial-derived secondary bile acids; 4) decreased thickness of the mucus layer adjacent to the intestinal epithelium with redistribution of mucus in the colon; and 5) increased serum levels of desaminotyrosine (DAT), a PAC-derived MM associated with immune modulation and resilience against virus-induced inflammation. Finally, we showed that PACs, are sufficient to increase intestinal abundance of A. muciniphila. Our data suggest PAC-induced alterations of the intestinal milieu promote metabolic resilience. To establish cause-effect relationships we propose to: 1) correlate urine/blood metabolites with gut bacterial strains/species using samples collected in longitudinal studies of GP-supplemented humans and PAC-treated mice with concomitant monitoring of metabolic and histological phenotypes in HFD- and low- fat diet (LFD)-fed murine hosts; 2) perform in vitro and germfree mouse studies to confirm relationship of specific bacteria isolate/consortia to specific MM; 3) investigate bioactivities of PAC-derived MM in cell-based assays related to glucose metabolism and the B29-monoassociated GF mouse model of HFD-induced MetS/T2D.